Trial of Selective Early Treatment of Patent Ductus Arteriosus with Ibuprofen.

BACKGROUND: The cyclooxygenase inhibitor ibuprofen may be used to treat patent ductus arteriosus (PDA) in preterm infants. Whether selective early treatment of large PDAs with ibuprofen would improve short-term outcomes is not known. METHODS: We conducted a multicenter, randomized, double-blind, placebo-controlled trial evaluating early treatment (≤72 hours after birth) with ibuprofen for a large PDA (diameter of ≥1.5 mm with pulsatile flow) in extremely preterm infants (born between 23 weeks 0 days' and 28 weeks 6 days' gestation). The primary outcome was a composite of death or moderate or severe bronchopulmonary dysplasia evaluated at 36 weeks of postmenstrual age. RESULTS: A total of 326 infants were assigned to receive ibuprofen and 327 to receive placebo; 324 and 322, respectively, had data available for outcome analyses. A primary-outcome event occurred in 220 of 318 infants (69.2%) in the ibuprofen group and 202 of 318 infants (63.5%) in the placebo group (adjusted risk ratio, 1.09; 95% confidence interval [CI], 0.98 to 1.20; P = 0.10). A total of 44 of 323 infants (13.6%) in the ibuprofen group and 33 of 321 infants (10.3%) in the placebo group died (adjusted risk ratio, 1.32; 95% CI, 0.92 to 1.90). Among the infants who survived to 36 weeks of postmenstrual age, moderate or severe bronchopulmonary dysplasia occurred in 176 of 274 (64.2%) in the ibuprofen group and 169 of 285 (59.3%) in the placebo group (adjusted risk ratio, 1.09; 95% CI, 0.96 to 1.23). Two unforeseeable serious adverse events occurred that were possibly related to ibuprofen. CONCLUSIONS: The risk of death or moderate or severe bronchopulmonary dysplasia at 36 weeks of postmenstrual age was not significantly lower among infants who received early treatment with ibuprofen than among those who received placebo. (Funded by the National Institute for Health Research Health Technology Assessment Programme; Baby-OSCAR ISRCTN Registry number, ISRCTN84264977.).

An Undergraduate Laboratory Exploring Mutational Mechanisms in Escherichia coli Based on the Luria-Delbrück Experiment.

Understanding the mechanism for DNA mutations is a key concept in most genetics and microbiology courses. In addition, understanding that most mutations occur prior to exposure to selection is an important yet often difficult concept for students to grasp. We developed an undergraduate laboratory activity on mutation mechanisms based on the classic experiment from Luria and Delbrück. The activity uses Escherichia coli as the model organism and the antibiotic streptomycin for selection. Students gain hands-on experience with an important experiment in genetics, and the laboratory contains an investigative component in having students calculate mutation rate for streptomycin resistance and in having the students design a follow-up experiment. E. coli has a knockout collection available, and we used a wild-type strain and a ΔmutS strain in the laboratory exercise. The ΔmutS strain is missing an enzyme in the mismatch repair pathway, and students calculate and compare the mutation rate and frequency for both the wild type and the knockout strain. Assessment of student learning showed that students had a significant gain in understanding of mutational mechanisms. An optional, additional experiment involving PCR and DNA sequencing of streptomycin-resistant mutants is also presented.

Draft Genome Sequences of Four Bacterial Species as Part of an Experiential Microbiology Project at SUNY Geneseo.

We report four draft genome sequences related to the genera Bacillus and Escherichia, recovered from surfaces associated with human interaction, and Sediminibacterium, recovered from an aquatic environment. This study was part of an undergraduate microbial bioinformatics course at the State University of New York at Geneseo.

Developmental stage influences chromosome segregation patterns and arrangement in the extremely polyploid, giant bacterium Epulopiscium sp. type B.

Few studies have described chromosomal dynamics in bacterial cells with more than two complete chromosome copies or described changes with respect to development in polyploid cells. We examined the arrangement of chromosomal loci in the very large, highly polyploid, uncultivated intestinal symbiont Epulopiscium sp. type B using fluorescent in situ hybridization. We found that in new offspring, chromosome replication origins (oriCs) are arranged in a three-dimensional array throughout the cytoplasm. As development progresses, most oriCs become peripherally located. Siblings within a mother cell have similar numbers of oriCs. When chromosome orientation was assessed in situ by labeling two chromosomal regions, no specific pattern was detected. The Epulopiscium genome codes for many of the conserved positional guide proteins used for chromosome segregation in bacteria. Based on this study, we present a model that conserved chromosomal maintenance proteins, combined with entropic demixing, provide the forces necessary for distributing oriCs. Without the positional regulation afforded by radial confinement, chromosomes are more randomly oriented in Epulopiscium than in most small rod-shaped cells. Furthermore, we suggest that the random orientation of individual chromosomes in large polyploid cells would not hamper reproductive success as it would in smaller cells with more limited genomic resources.

Identification of Allorecognition Loci in Neurospora crassa by Genomics and Evolutionary Approaches.

Understanding the genetic and molecular bases of the ability to distinguish self from nonself (allorecognition) and mechanisms underlying evolution of allorecognition systems is an important endeavor for understanding cases where it becomes dysfunctional, such as in autoimmune disorders. In filamentous fungi, allorecognition can result in vegetative or heterokaryon incompatibility, which is a type of programmed cell death that occurs following fusion of genetically different cells. Allorecognition is genetically controlled by het loci, with coexpression of any combination of incompatible alleles triggering vegetative incompatibility. Herein, we identified, characterized, and inferred the evolutionary history of candidate het loci in the filamentous fungus Neurospora crassa. As characterized het loci encode proteins carrying an HET domain, we annotated HET domain genes in 25 isolates from a natural population along with the N. crassa reference genome using resequencing data. Because allorecognition systems can be affected by frequency-dependent selection favoring rare alleles (i.e., balancing selection), we mined resequencing data for HET domain loci whose alleles displayed elevated levels of variability, excess of intermediate frequency alleles, and deep gene genealogies. From these analyses, 34 HET domain loci were identified as likely to be under balancing selection. Using transformation, incompatibility assays and genetic analyses, we determined that one of these candidates functioned as a het locus (het-e). The het-e locus has three divergent allelic groups that showed signatures of positive selection, intra- and intergroup recombination, and trans-species polymorphism. Our findings represent a compelling case of balancing selection functioning on multiple alleles across multiple loci potentially involved in allorecognition.

Sporulation in Bacteria: Beyond the Standard Model.

Endospore formation follows a complex, highly regulated developmental pathway that occurs in a broad range of Firmicutes. Although Bacillus subtilis has served as a powerful model system to study the morphological, biochemical, and genetic determinants of sporulation, fundamental aspects of the program remain mysterious for other genera. For example, it is entirely unknown how most lineages within the Firmicutes regulate entry into sporulation. Additionally, little is known about how the sporulation pathway has evolved novel spore forms and reproductive schemes. Here, we describe endospore and internal offspring development in diverse Firmicutes and outline progress in characterizing these programs. Moreover, comparative genomics studies are identifying highly conserved sporulation genes, and predictions of sporulation potential in new isolates and uncultured bacteria can be made from these data. One surprising outcome of these comparative studies is that core regulatory and some structural aspects of the program appear to be universally conserved. This suggests that a robust and sophisticated developmental framework was already in place in the last common ancestor of all extant Firmicutes that produce internal offspring or endospores. The study of sporulation in model systems beyond B. subtilis will continue to provide key information on the flexibility of the program and provide insights into how changes in this developmental course may confer advantages to cells in diverse environments.

Diversification of a protein kinase cascade: IME-2 is involved in nonself recognition and programmed cell death in Neurospora crassa.

Kinase cascades and the modification of proteins by phosphorylation are major mechanisms for cell signaling and communication, and evolution of these signaling pathways can contribute to new developmental or environmental response pathways. The Saccharomyces cerevisiae kinase Ime2 has been well characterized for its role in meiosis. However, recent studies have revealed alternative functions for Ime2 in both S. cerevisiae and other fungi. In the filamentous fungus Neurospora crassa, the IME2 homolog (ime-2) is not required for meiosis. Here we determine that ime-2 interacts genetically with a transcription factor vib-1 during nonself recognition and programmed cell death (PCD). Mutations in vib-1 (Δvib-1) suppress PCD due to nonself recognition events; however, a Δvib-1 Δime-2 mutant restored wild-type levels of cell death. A role for ime-2 in the post-translational processing and localization of a mitochondrial matrix protein was identified, which may implicate mitochondria in N. crassa nonself recognition and PCD. Further, Δvib-1 strains do not produce extracellular proteases, but protease secretion reverted to near wild-type levels in a Δvib-1 Δime-2 strain. Mass spectrometry analysis revealed that the VIB-1 protein is phosphorylated at several sites, including a site that matches the IME-2 consensus. The genetic and biochemical data for ime-2 and vib-1 indicate that IME-2 is a negative regulator of VIB-1 and suggest parallel negative regulation by IME-2 of a cell death pathway in N. crassa that functions in concert with the VIB-1 cell death pathway. Thus, IME2 kinase function has evolved following the divergence of S. cerevisiae and N. crassa and provides insight into the evolution of kinases and their regulatory targets.

Meiotic regulators Ndt80 and ime2 have different roles in Saccharomyces and Neurospora.

Meiosis is a highly regulated process in eukaryotic species. The filamentous fungus Neurospora crassa has been shown to be missing homologs of a number of meiotic initiation genes conserved in Saccharomyces cerevisiae, but has three homologs of the well-characterized middle meiotic transcriptional regulator NDT80. In this study, we evaluated the role of all three NDT80 homologs in the formation of female reproductive structures, sexual development, and meiosis. We found that none of the NDT80 homologs were required for meiosis and that even the triple mutant was unaffected. However, strains containing mutations in NCU09915 (fsd-1) were defective in female sexual development and ascospore maturation. vib-1 was a major regulator of protoperithecial development in N. crassa, and double mutants carrying deletions of both vib-1 (NCU03725) and fsd-1 exhibited a synergistic effect on the timing of female reproductive structure (protoperithecia) formation. We further evaluated the role of the N. crassa homolog of IME2, a kinase involved in initiation of meiosis in S. cerevisiae. Strains containing mutations in ime-2 showed unregulated development of protoperithecia. Genetic analysis indicated that mutations in vib-1 were epistatic to ime-2, suggesting that IME-2 may negatively regulate VIB-1 activity. Our data indicate that the IME2/NDT80 pathway is not involved in meiosis in N. crassa, but rather regulates the formation of female reproductive structures.

Achieving routine sub 30 minute door-to-balloon times in a high volume 24/7 primary angioplasty center with autonomous ambulance diagnosis and immediate catheter laboratory access.

BACKGROUND: In primary angioplasty (primary percutaneous coronary intervention [PPCI]) for acute myocardial infarction, institutional logistical delays can increase door-to-balloon times, resulting in increased mortality. METHODS: We moved from a thrombolysis (TL) service to 24/7 PPCI for direct access and interhospital transfer in April 2004. Using autonomous ambulance diagnosis with open access to the myocardial infarction center catheter laboratory, we compared reperfusion times and clinical outcomes for the final 2 years of TL with the first 3 years of PPCI. RESULTS: Comparison was made between TL (2002-2004, n = 185) and PPCI (2004-2007, n = 704); all times are medians in minutes (interquartile range): for TL, symptom to needle 153 (85-225), call to needle 58 (49-73), first professional contact (FPC) to needle 47 (39-63), door to needle 18 (12-30) (mortality: 7.6% at 30 days, 9.2% at 1 year); for interhospital transfer PPCI (n = 227), symptom to balloon 226 (175-350), call to balloon 135 (117-188), FPC to balloon 121 (102-166), first door-to-balloon 100 (80-142) (mortality: 7.0% at 30 days, 12.3% at 1 year); for direct-access PPCI (n = 477), symptom to balloon 142 (101-238), call to balloon 79 (70-93), FPC to balloon 69 (59-82), door to balloon 20 (16-29) (mortality: 4.6% at 30 days, 8.6% at 1 year). There was no difference between direct-access PPCI and TL times for symptom to needle/balloon. Direct-access PPCI was significantly quicker for the group than in-hospital thrombolysis for door to needle/balloon times due to the lack of any long wait patients (P < .001). CONCLUSIONS: Interhospital transfer remains slow even with rapid institutional door-to-balloon times. With autonomous ambulance diagnosis and open access direct to the catheter laboratory, a median door-to-balloon time of <30 minutes day and night was achieved, and >95% of patients were reperfused within 1 hour.

Transcriptional profiling and functional analysis of heterokaryon incompatibility in Neurospora crassa reveals that reactive oxygen species, but not metacaspases, are associated with programmed cell death.

Heterokaryon incompatibility (HI) is a nonself recognition phenomenon occurring in filamentous fungi that is important for limiting resource plundering and restricting viral transfer between strains. Nonself recognition and HI occurs during hyphal fusion between strains that differ at het loci. If two strains undergo hyphal fusion, but differ in allelic specificity at a het locus, the fusion cell is compartmentalized and undergoes a rapid programmed cell death (PCD). Incompatible heterokaryons show a macroscopic phenotype of slow growth and diminished conidiation, and a microscopic phenotype of hyphal compartmentation and cell death. To understand processes associated with HI and PCD, we used whole-genome microarrays for Neurospora crassa to assess transcriptional differences associated with induction of HI mediated by differences in het-c pin-c haplotype. Our data show that HI is a dynamic and transcriptionally active process. The production of reactive oxygen species is implicated in the execution of HI and PCD in N. crassa, as are several genes involved in phosphatidylinositol and calcium signalling pathways. However, genes encoding mammalian homologues of caspases or apoptosis-inducing factor (AIF) are not required for HI or programmed cell death. These data indicate that PCD during HI occurs via a novel and possibly fungal-specific mechanism, making this pathway an attractive drug target for control of fungal infections.